JNK1 physically interacts with WW domain-containing oxidoreductase (WOX1) and inhibits WOX1-mediated apoptosis.

Transient activation of c-Jun N-terminal kinase (JNK) promotes cell survival, whereas persistent JNK activation induces apoptosis. Bovine testicular hyaluronidase PH-20 activates JNK1 and protects L929 fibroblasts from staurosporine-mediated cell death. PH-20 also induces the expression of a p53-interacting WW domain-containing oxidoreductase (WOX1, also known as WWOX or FOR) in these cells. WOX1 enhances the cytotoxic function of tumor necrosis factor and mediates apoptosis synergistically with p53. Thus, the activated JNK1 is likely to counteract WOX1 in mediating apoptosis. Here it is demonstrated that ectopic JNK1 inhibited WOX1-mediated apoptosis of L929 fibroblasts, monocytic U937 cells, and other cell types. Also, JNK1 blocked WOX1 prevention of cell cycle progression. By stimulating cells with anisomycin or UV light, JNK1 became activated, and WOX1 was phosphorylated at Tyr(33). The activated JNK1 physically interacted with the phosphorylated WOX1, as determined by co-immunoprecipitation. Alteration of Tyr(33) to Arg(33) in WOX1 abrogated its binding interaction with JNK1 and its activity in mediating cell death, indicating that Tyr(33) phosphorylation is needed to activate WOX1. A dominant negative WOX1 was developed and shown to block p53-mediated apoptosis and anisomycin-mediated WOX1 phosphorylation but could not inhibit JNK1 activation. This mutant protein bound p53 but could not interact with JNK1, as determined in yeast two-hybrid analysis. Taken together, phosphorylation of JNK1 and WOX1 is necessary for their physical interaction and functional antagonism.

The WWOX gene coding for human WOX1 is mapped to a fragile site on the chromosome ch16q (for reviews, see Refs. 2 and 3). High frequency of loss of heterozygosity of this chromosomal region has been shown in breast (6) and prostate cancers (7,8). This chromosome region appears to confer suppression of the metastatic activity in a prostatic cancer cell line (9). Both in vitro and in vivo experiments showed that WOX1 could act as a tumor suppressor (10). WOX1 (46 kDa) possesses two N-terminal WW domains (containing conserved tryptophan residues), a nuclear localization sequence (NLS) and a C-terminal short-chain alcohol dehydrogenase (ADH) domain (1). The human WWOX gene has nine exons. At least seven alternatively spliced human WOX protein variants have been found. The ADH domain region, which is encoded by exons 4 -8, is frequently deleted or alternatively spliced in the variants. In contrast, the WW domain region is rarely deleted, and its amino acid sequence is highly conserved among humans, mice, and rats. However, in Drosophila, the sequence of the N-terminal WW domain region is altered (11).
A portion of cytosolic WOX1 is located in the mitochondria, and the mitochondrial targeting sequence was mapped within the ADH domain (1). Induction of mitochondrial permeability transition by TNF, staurosporine, and atractyloside results in WOX1 release from the mitochondria and subsequent nuclear translocation (1,2). c-Jun N-terminal kinase (JNK) is involved in cellular stress response and has been implicated in both cell growth and apoptosis (for a review, see Ref. 12). JNK1 is involved in signaling pathways that initiate cell cycle checkpoints and cell cycle progression (13). In contrast, inhibition of the JNK signaling pathway prevents death of trophic factor-deprived neurons (14). Persistent activation of JNK1 causes apoptosis (15). However, early activation of JNK1 is essential for protecting cells against TNF-mediated apoptosis (16,17). Transient activation of JNK by growth factors promotes survival of human thyrocytes (18). Notably, hyaluronidase activation of JNK1 and JNK2 protects L929 fibroblasts from staurosporine-mediated cell death (19).
In this study, we demonstrated that anisomycin and UV light activated JNK1 and induced WOX1 phosphorylation in Tyr 33 , and both proteins physically interacted. Ectopic JNK1 inhibited WOX1-mediated apoptosis of L929 fibroblasts, monocytic U937 cells, and other tested cells. Also, WOX1 prevention of cell cycle progression was abolished by JNK1. The mecha-nisms of JNK1 and WOX1 binding interaction and the functional significance of this interaction were investigated.

EXPERIMENTAL PROCEDURES
Cell Lines-Cell lines used in these studies were murine L929 fibroblasts, human monocytic U937 cells, human prostate Du145 cells, and human neuroblastoma SK-N-SH cells. These cells were grown according to the instructions of the ATCC (Manassas, VA).
cDNA Expression Constructs-Expression constructs for the murine wild type Wox1 cDNA (sense and antisense directions) and a nuclear localization mutant Wox1mtNLS were established as previously described (1). These constructs were made in pEGFP-C1 vector (Clontech, Palo Alto, CA). The expressed proteins were tagged with an N-terminal enhanced green fluorescent protein.
Cell Cycle Analysis and Cell Death Assays-Where indicated, L929 cells were electroporated with the wild type or DN-JNK constructs (20 g of DNA/3 ϫ 10 6 cells; 200 V and 50 ms; BTX ECM 830 Square Wave Electroporator; Genetronics, San Diego, CA). Electroporation caused immediate cell death (or necrosis) by ϳ20 -40%. These electroporated cells were seeded and cultured on 60-mm Petri dishes (Corning Glass) overnight. The culture supernatants were discarded to remove dead cells. The cells were fed with fresh medium (RPMI 1640 plus 10% fetal bovine serum) and then treated with or without bovine testicular hyaluronidase (100 units/ml; Sigma) for 24 h. The cell layers, reaching 80 -90% confluence in 48 h, were gently washed once with phosphatebuffered saline, treated with 70% ethanol, harvested by repeat pipetting, precipitated by centrifugation (500 ϫ g), and then stained with propidium iodide. The cellular DNA content was analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ).
In some experiments, L929 and other cells were electroporated with WOX1 and/or JNK1 constructs and cultured for 24 h (in eight replicates). The extent of cell death was examined by crystal violet staining (1) or by Promega's MTS proliferation assay (Promega, Madison, WI).
Antibodies, Western Blotting, and Co-immunoprecipitation-Antibody against the N-terminal amino acid sequence of WOX1 was generated as previously described (1). Commercial antibodies used in the Western blotting were against JNK1 and a phospho-JNK1 peptide (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Co-immunoprecipitation was performed as previously described (1,20). Briefly, U937, L929, or SK-N-SH cells were treated with anisomycin (100 M) for various indicated times to activate JNK1. The cytosolic and nuclear fractions of these cells were prepared using the NE-PER nuclear and cytoplasmic extraction reagents (Pierce). Endogenous JNK1 was precipitated by the specific anti-JNK1 antibody (Santa Cruz Biotechnology) and protein A-agarose beads (Pierce), followed by separation in SDS-PAGE and detection by immunostaining. Co-presence of WOX1 in the precipitates was examined similarly by immunostaining. Alternatively, immunoprecipitation was performed using anti-WOX1 or p53 (Santa Cruz Biotechnology) antibody.
Generation of Antibody against a Phospho-Tyr 33 WOX1 Peptide-Generation of antibody against synthetic peptides in rabbits was performed as described (1). A synthetic peptide NH 2 -CKDGWVpYY-ANHTEEKT-COOH, with tyrosine 33 phosphorylation (pY), was made (Genemed Synthesis, South San Francisco, CA). A control peptide without phosphorylation was made also. The N-terminal cysteine was added to allow the peptide to covalently conjugate to keyhole limpet hemocyanin for immunization or to SulfoLink beads for antibody purification (Pierce). The peptide sequence is located at the first WW domain of murine WOX1 (amino acids 28 -38). Antibody generation was performed using the EZ antibody production and purification kit (Pierce). The antiserum was preadsorbed with a column covalently conjugated with the control peptide, and then the antibody was purified by affinity chromatography using a column immobilized with the synthetic phosphopeptide.
Yeast Two-hybrid Interactions and Constructs-The Ras rescuebased CytoTrap yeast two-hybrid system (Stratagene, La Jolla, CA) for protein binding interaction was used (1,20). Briefly, binding of a cytosolic Sos-tagged bait protein to a cell membrane-anchored target protein (tagged with a myristoylation signal) results in activation of the Ras signaling pathway in yeast. Activation of the Ras signaling pathway allows mutant yeast Cdc25H to grow in 37°C using a selective agarose plate containing galactose. Without binding, yeast cells fail to grow at 37°C. The human JNK1 cDNA was constructed in a pMyr vector and utilized as target. The bait constructs in a pSos vector for binding interactions with JNK1 were murine Wox1 (1), human WOX3 (1), and the first WW domain of murine Wox1 (1). Additional constructs for control experiments were human p53 in pMyr, MafB in both pMyr and pSos, collagenase in pSos, and lamin C in pMyr (1,20).
Statistical Analysis and Data Presentation-Where indicated, Student's t tests were performed to analyze the experimental data (using the Microsoft Excel program). All experiments indicated above were performed 2-5 times. In most cases, a representative set of data is shown.
To examine the possible role of JNK1 in the PH-20-mediated growth regulation, L929 cells were electroporated with a wild type human JNK1 or a dominant negative JNK1 construct. The cells were cultured overnight, exposed to PH-20 for 24 h, and then subjected to cell cycle analysis by flow cytometry. In controls, the cells were electroporated with nothing (or medium only) and treated similarly. The results showed that ectopic wild type JNK1 did not induce apoptosis or death of L929 cells (Fig. 2). Exposure of these JNK1-expressing cells to PH-20 could not induce apoptosis (Fig. 2). In contrast, dominantnegative JNK1 significantly induced L929 cell death (56.4%), and PH-20 did not significantly increase the extent of cell death (60.9%) (Fig. 2). In comparison, ectopic human wild-type JNK2 and JNK3 mediated apoptosis or death of L929 cells by ϳ30%, and PH-20 limitedly increased the cell death (ϳ40%) (Fig. 2). In contrast, dominant negative JNK2 and JNK3 significantly mediated cell death up to 60 -70%, and PH-20 slightly increased the cell death ( Fig. 2).
JNK1 Inhibition of WOX1-mediated Cell Death-PH-20 induces the expression of proapoptotic p53 and WOX1 in L929 cells, thereby increasing cellular sensitivity to the cytotoxic effect of TNF (1). Nonetheless, PH-20 also activates JNK1 in these cells (19). Here, we examined whether JNK1 inhibited WOX1-mediated apoptosis of L929 cells. In agreement with our previous observations (1), electroporation of L929 cells with a wild-type WOX1 expression construct (tagged with enhanced green fluorescent protein) resulted in cell death (Fig. 3). The extent of cell death was determined by the MTS assay. Similar results were observed by staining the cells with crystal violet (data not shown). The extent of WOX1 protein expression was ϳ60 -75% in L929 cells, as determined by counting cells under fluorescence microscopy.
When L929 cells were cotransfected with both wild type JNK1 and WOX1 constructs, and JNK1 blocked WOX1-mediated cell death (Fig. 3). In contrast, WOX1mtNLS, which possesses a mutated sequence at the NLS, had a significantly reduced activity in mediating cell death (Fig. 3). When in combination, JNK1 and WOX1mtNLS could not induce cell death (Fig. 3). The mutation in WOX1mtNLS prevents nuclear translocation of this protein (1). In agreement with our previous observations (1), expression of antisense WOX1 mRNA in L929 cells failed to result in cell death, and JNK1 had no effect in mediating cell death during cotransfection (Fig. 3). Together, WOX1 mediates apoptosis in the nucleus, and JNK1 blocks the WOX1-mediated cell death at the nuclear level. L929 cells were transfected with JNK1 and/or various WOX1 constructs by electroporation and cultured for 24 h. The extent of cell death was measured by the MTS assay. Wild type WOX1 mediated L929 cell death (ϳ40%), which was blocked by JNK1 (ϳ20% death) (p Ͻ 0.0001, n ϭ 8, Student's t test). JNK1 alone slightly increased cell proliferation (0.001 Ͻ p Ͻ 0.05, n ϭ 8, Student's t test). In contrast, WOX1mtNLS, which has a mutated sequence at the NLS, had a significantly reduced activity in inducing cell death (ϳ70% reduction). This mutant failed to undergo nuclear translocation (1). When in combination, JNK1 and WOX1mtNLS could not significantly mediate cell death (p Ͼ 0.1, n ϭ 8, Student's t test). Expression of antisense WOX1 mRNA in L929 cells could not mediate cell death. JNK1 and the antisense WOX1, in combination, could not induce cell death either (p Ͼ 0.1, n ϭ 8, Student's t test). Experiments were done in eight replicates (mean Ϯ S.D.). A representative set of data from two experiments is shown. In controls, the cells were electroporated with nothing or medium (mock) and treated similarly (A). The wild type JNK1 did not induce apoptosis of L929 cells. In contrast, dominant-negative JNK1 significantly induced L929 cell death, and hyaluronidase (HAase) slightly increased the extent of cell death. In comparison, wild type JNK2 and JNK3 mediated L929 cell death by ϳ30%, and the dominant negatives of these proteins significantly caused cell death (ϳ60 -70%). A representative set of data from two experiments is shown. JNK1 Blocks WOX1-mediated Inhibition of Cell Cycle Progression in U937 Cells-To further verify the above observations, we examined the ability of wild type JNK1 in blocking apoptosis of monocytic U937 cells by WOX1. U937 cells were electroporated with various amounts of WOX1 in the presence or absence of wild type JNK1. The cells were cultured 48 h and subjected to cell cycle analysis by flow cytometry. The results showed that WOX1-mediated apoptosis was suppressed by JNK1 (see the sub-G 0 /G 1 phase) (Fig. 4). Additionally, WOX1mediated reduction of cellular distribution in the G 0 /G 1 , S, and G 2 /M phases was reversed by JNK1 (Fig. 4). Thus, our data strongly support the protective role of JNK1 in blocking WOX1mediated cell death.
WOX1 Physically Interacts with JNK1-We investigated whether JNK1 physically interacts with WOX1, thereby blocking WOX1-mediated apoptosis. As determined by yeast twohybrid analysis (1,20), the wild type human JNK1 interacted with murine WOX1 and human WOX3 in vivo, as evidenced by the growth of mutant Cdc25H yeast at 37°C (Fig. 5). Interestingly, the WOX3/JNK1 binding interaction promoted a strong yeast cell growth, compared with that of the WOX1/JNK1 interaction (Fig. 5). Human WOX3 has a deletion of its ADH domain at the C-terminus, whereas its N-terminal WW domain region is highly homologous to that of murine WOX1 (Fig. 5) (1). The first WW domain also interacted with JNK1 (Fig. 5). These results suggest that the first WW domain is responsible for binding to JNK1. A conserved phosphorylation site at the first WW domain, Tyr 33 , was then altered to arginine in WOX3. This mutant, designated WOX3(Y33R), could not interact with JNK1 (Fig. 5). The observations further suggest that phosphorylation at Tyr 33 is essential for WOX1 and JNK1 binding interaction.
In positive control experiments, the binding interactions between p53 and WOX1 (1) and the self-binding interaction of MafB were demonstrated (Fig. 5). In negative controls, no binding interactions were observed when testing empty pSos vector versus empty pMyr vector, or collagenase versus lamin C (Fig. 5).
Anisomycin Activates JNK1 and Mediates Tyr 33 Phosphorylation in WOX1 and Their Binding Interactions-To further verify the above observations, U937 cells were treated with anisomycin to activate JNK1. Anisomycin is a potent activator of JNK1 (17). In resting cells, no JNK1 and WOX1 binding interaction was observed, as determined by co-immunoprecipitation using specific anti-JNK1 antibody (Fig. 6A). Upon stimulation of cells with anisomycin for 30 min, expression of JNK1 was increased, and a portion of JNK1 interacted with WOX1 (Fig. 6A).
Similarly, exposure of SK-N-SH cells to anisomycin for 30 min also resulted in an increased binding interaction between JNK1 and WOX1, as determined using anti-WOX1 antibody in co-immunoprecipitation (Fig. 6B). Similar results were observed when L929 cells were treated with anisomycin, or the above cells were exposed to UV light (data not shown).
To further verify Tyr 33 phosphorylation in WOX1, we synthesized a peptide containing phospho-Tyr 33 for immunization in rabbits. Anisomycin rapidly increased Tyr 33 phosphorylation in WOX1, as well as JNK1 phosphorylation, in SK-N-SH cells (Fig. 7A). Similar results were observed using L929 and U937 cells (data not shown).
Exposure of L929 cells to UV light, followed by growing for 1 h, resulted in increased p53 expression (Fig. 7B). p53 is known to interact with WOX1 (1) and JNK1 (21). Co-immunoprecipitation of p53 with a specific antibody resulted in the  (1,20). In positive controls, the binding interactions between p53 and WOX1 (1) and the self-binding interaction of MafB are shown, as evidenced by the growth of mutant Cdc25H yeast at 37°C in the presence of galactose. In negative controls, no binding interactions were observed when testing empty pSos vector versus empty pMyr vector or collagenase versus lamin C. The wild type human JNK1 physically interacted with murine WOX1 and human WOX3. Human WOX3 has a deletion of its ADH domain at the C-terminus, whereas its N-terminal WW domain region is highly homologous to that of murine WOX1 (1). The first WW domain interacted with JNK1. Alteration of a conserved phosphorylation site at Tyr 33 to Arg 33 at the first WW domain in WOX3 was performed. This mutant, designated WOX3(Y33R), could not bind JNK1. A representative set of data from three experiments is shown.
Dominant Negative WOX1 Abolishes WOX1 Phosphorylation and p53 Apoptosis but Not JNK1 Activation-We developed a dominant-negative WOX1. Lys 28 and Asp 29 in Wox1 were mutated to Thr 28 and Val 29 , respectively. These residues are located at the first WW domain. In agreement with our previous observations (1), p53 and WOX1 mediated U937 cell death in a synergistic manner, and antisense WOX1 abolished p53-mediated cell death (Fig. 8A). The mutated full-length WOX1 (WOX1mt) abolished p53 apoptosis (Fig. 8A). Similarly, the mutated WW domain region (WOX1wwmt) also abolished p53 apoptosis (Fig. 8A). These data support the dominant negative effect of the generated WOX1 mutant.
Anisomycin-mediated WOX1 phosphorylation in Tyr 33 was abolished by the dominant negative WOX1 (Fig. 8B). SK-N-SH cells were transfected with a GFP-tagged WOX1wwmt or a control GFP construct by electroporation. The cells were cultured for 48 h and then treated with anisomycin for 30 min. The dominant negative WOX1wwmt abolished anisomycin-mediated WOX1 phosphorylation in Tyr 33 (Fig. 8B). However, WOX1wwmt failed to inhibit anisomycin-induced JNK1 activation (Fig. 8B).
By yeast two-hybrid analysis, both WOX1mt and WOX1wwmt physically interacted with p53, whereas WOX1wwmt could not bind JNK1 (Fig. 8C). Apparently, these dominant negative WOX1 proteins have an altered conformation, and inhibition of p53 apoptosis by the dominant negatives is due to a direct binding interaction. In contrast, these dominant negatives failed to bind and prevent JNK1 activation.

Tyr 33 Phosphorylation Is Necessary for WOX1 Apoptotic
Function-Overexpression of the WW domain region of WOX1, designated WOX1ww, induces cell death (1). Alteration of Tyr 33 to Arg 33 was performed in WOX1ww, and the resulting mutant, WOX1ww(Y33R), had a significantly reduced activity (ϳ50% decrease) in mediating death of L929 and Du145 cells (Fig. 9). The findings were also observed using U937, SK-N-SH, and other types of cells (data not shown). JNK1 significantly blocked WOX1ww-mediated death of L929 and Du145 cells by 30 -50% (Fig. 9). Interestingly, JNK1 increased the apoptotic function of WOX1ww(Y33R) mutant in L929 but not in Du145 cells (Fig. 9).

DISCUSSION
In this study, we demonstrated that stress stimuli such as anisomycin and UV light mediated WOX1 phosphorylation at Tyr 4 . The phosphorylation is essential for functional activation of WOX1 in mediating apoptosis. Alteration of Tyr 33 to Arg 33 reduced the WOX1 activity. In agreement with our previous observations (1), WOX1-mediated apoptosis occurs at the nuclear level. Mutation of the nuclear localization signal reduced the WOX1 apoptotic function.
We determined that ectopic JNK1 counteracted WOX1-mediated apoptosis, as well as cell cycle progression. By stimulating cells with anisomycin, JNK1 became activated and physically interacted with the Tyr 33 -phosphorylated WOX1, as determined by co-immunoprecipitation. A dominant negative WOX1 blocked p53-mediated apoptosis and anisomycin-mediated WOX1 phosphorylation but failed to inhibit JNK1 activation. This dominant negative WOX1 bound p53 but could not interact with JNK1, as determined in yeast two-hybrid analy-FIG. 6. JNK1 and WOX1 binding interaction in anisomycintreated cells. A, monocytic U937 cells were treated with anisomycin (100 M) to activate JNK1 (17). The cells were then processed for immunoprecipitation (IP) using specific anti-JNK1 antibody. The presence of a cytosolic JNK1 and WOX1 complex was observed in cells treated with anisomycin for 30 min. In negative controls, no JNK1 was observed when protein A-agarose beads alone were used in the precipitation. IgH, IgG heavy chain; Pre-IP, one-tenth of the total cytosolic and nuclear proteins (ϳ50 g) were loaded onto SDS-PAGE for Western blotting analysis. B, similar results were observed by exposure of SK-N-SH neuroblastoma cells to anisomycin (100 M) for 30 min, followed by co-immunoprecipitation using anti-WOX1 antibody. Representative data from three experiments are shown.

FIG. 7. Anisomycin and UV light mediated Tyr33 phosphorylation in WOX1.
A, SK-N-SH cells were stimulated with anisomycin (100 M) for various indicated times. Activation (or phosphorylation) of JNK1 and phosphorylation of WOX1 were observed. B, L929 cells were exposed to UV light and then cultured for 1 h, followed by processing co-immunoprecipitation using specific anti-p53 antibody. UV induced p53 expression and its binding interaction with JNK1 and the Tyr 33phosphorylated WOX1. IgH, IgG heavy chain. Representative data from three experiments are shown.
sis. Together, these observations indicate that a physical contact between activated or phosphorylated WOX1 and JNK1 is essential for their functional antagonism. How JNK1 inhibits WOX1 function remains to be determined.
The upstream kinase that phosphorylates WOX1 is unknown. Nonetheless, we do not exclude the possibility that activated JNK1 binds WOX1 and then phosphorylates WOX1. Alternatively, an unknown kinase phosphorylates WOX1 in response to stress stimuli. The phosphorylated WOX1 then interacts with JNK1.
The dominant negative WOX1 bound p53 and blocked p53 apoptosis. Also, this mutant protein inhibited WOX1 phosphorylation, suggesting its possible interaction with the wild type WOX1. This potential binding interaction remains to be determined by co-immunoprecipitation and yeast two-hybrid analysis.
An additional conserved phosphorylation site in WOX1 is Tyr 61 . Alteration of this residue to Arg 61 results in abrogation of the binding interaction between WOX1 and p53 in yeast. 2 Thus, it is likely that both Tyr 33 and Tyr 61 in WOX1 are phosphorylated when cells are exposed to stress stimuli. Functional significance of Tyr 61 phosphorylation in WOX1 is being determined in this laboratory.
The functional property of WOX3 in vivo and in vitro is unknown. Whether WOX3 expression is increased during stress response is unknown. By RT-PCR and Northern blot, Driouch demonstrated that about 50% of breast tumors have overexpressed WOX1 and WOX3 (22). WOX1 appears to have a tumor suppressor function (10,11). Presumably, the overexpressed WOX3 enhances cancer growth by neutralizing the WOX1 function.
In addition to activate JNK1, anisomycin inhibits protein synthesis and mediates apoptosis (23,24). Anisomycin-mediated apoptosis of leukemia HL-60 cells depends upon JNK1 (24). Most interestingly, ansiomycin-activated JNK1 phosphorylates Bcl-2, thereby increasing cell survival (25). Previously, we found that when L929 cells were exposed to anisomycin for less than 1 h, these cells became resistant to TNF cytotoxicity (17). We determined that IB␣, the inhibitor of NF-B, maintains the basal level of JNK1 activation and regulates JNK1mediated TNF resistance (17). However, whether this protective event is related to JNK1-mediated Bcl-2 phosphorylation remains to be determined.
Hyaluronidase PH-20 activates JNK1, and the activation is necessary to prevent L929 cell death by various anticancer drugs such as staurosporine, daunorubicin, and doxorubicin (19). In contrast, PH-20 increases L929 cell sensitivity to TNF cytotoxicity by increasing the expression of WOX1 (1). Apparently, different mechanisms are involved in the regulation of cellular sensitivity to TNF and anticancer drugs. Indeed, we determined that ectopic JNK1 and exogenous PH-20 act synergistically in blocking L929 cell death by WOX1 in the presence of staurosporine, daunorubicin, or doxorubicin. 2 We believe that PH-20 activates both endogenous and ectopic JNK1 that block the cell death. Whether JNK1 mediates Bcl-2 phosphorylation in these tested cells is unknown.
The hyaluronidase HYAL1 gene, also known as LUCA1, is a candidate tumor suppressor (for a review, see Ref. 26). Inactivation of the HYAL1 gene by aberrant splicing of pre-mRNA has been shown in head and neck squamous cell carcinomas (27). Nonetheless, overexpressed hyaluronidases are frequently associated with metastatic cancers (28 -30). Also, plasma HYAL1 is capable of promoting tumor cell cycling (31). PH-20 enhances cell proliferation (4). Here, we demonstrated that PH-20 increased cell cycle progression in L929 cells. PH-20-activated JNK1 is essential for cell survival, since dominant negative JNK1 mediated apoptosis in L929 cells.
UV light up-regulates and activates p53 (32) and also activates JNK1 (12). Co-immunoprecipitation of the anisomycintreated cells with anti-p53 resulted in the presence of p53, JNK1, and WOX1 in the precipitate. The observation suggests the presence of a p53⅐WOX1⅐JNK1 complex during stress response. Most recently, we determined that a portion of cytosolic IB␣ binds p53 in resting cells and the complex dissociates in response to apoptotic stress, hypoxia, DNA damage, and tumor growth factor-␤1-mediated growth suppression (20). The dissociation allows p53 nuclear translocation. Alternatively, when dissociated from IB␣, p53 may complex with WOX1 and JNK1.
Although transient JNK1 activation provides a signal for cell 2 N.-S. Chang, J. Doherty, and A. Ensign, unpublished results.
FIG. 8. Dominant negative WOX1 abolishes WOX1 phosphorylation and p53 apoptosis but not JNK1 activation. A, a dominantnegative WOX1 was developed by mutating Lys 28 and Asp 29 to Thr 28 and Val 29 , respectively. U937 cells were electroporated with WOX1 expression constructs or nothing (null) in the presence or absence of a p53 expression construct. The cells were harvested 48 h postelectroporation, and DNA fragmentation was analyzed by agarose gel electrophoresis. p53-mediated cell death was blocked by the mutated fulllength WOX1 (WOX1mt), the mutated WW domain region (WOX1wwmt), and antisense WOX1 (WOX1as). The relative increase in DNA fragmentation (versus controls) was analyzed by the NIH Image program. The data represent average from two experiments. B, SK-N-SH cells were transfected with a GFP-tagged WOX1wwmt or a control GFP construct by electroporation, cultured for 48 h, and then treated with anisomycin for 30 min. WOX1wwmt abolished anisomycinmediated WOX1 phosphorylation in Tyr 33 . However, WOX1wwmt failed to inhibit anisomycin-induced JNK1 activation. C, both WOX1mt and WOX1wwmt physically interacted with p53, as determined in yeast. WOX1ww interacted with JNK1, whereas WOX1wwmt could not bind JNK1. A representative set of data from 2-4 experiments is shown in B and C.
survival (13, 16 -19), a persistent JNK1 activation can mediate cell death (14,15). How the antiapoptotic JNK1 turns into apoptotic is not known. It may be related to the status of JNK1 phosphorylation, or it may be associated with JNK1-regulated gene expression at different stages of cell differentiation or stress. We believe that at a certain stage of apoptosis, the apoptotic JNK1 may act synergistically with WOX1 in facilitating cell death.
In transient expression, both wild type JNK2 and JNK3 mediated L929 cell death, yet the dominant negatives greatly increased the cell death. The likely explanation is that the kinetics of activation of JNK isoforms is different, and their functional roles may be different also. For example, JNK1 and JNK2 have distinct roles in antiviral immunity, in which JNK1 is involved in survival of activated T cells during immune responses, and JNK2 plays a role in control of CD8(ϩ) T cell expansion in vivo (33). Similarly, JNK1 and JNK2 play distinct roles in controlling the activation of CD8(ϩ) T cells (34). Also, JNK1-deficient cells resist UV-mediated apoptosis, whereas JNK2-deficient cells have increased sensitivity to UV irradiation (35). JNKs are widely distributed in the nervous system (12), and JNK3 is involved not only in neuronal development but also apoptosis of neuronal cells (36).
L929 cells express both JNK1 and JNK2 (17,19). Whether JNK3 is expressed in L929 cells is unknown. Dominant negative JNK1 mediated L929 cell apoptosis, suggesting that JNK1 is essential for the survival of these cells. Inhibition of JNK2 and JNK3 functions by dominant negatives induced cell death, suggesting that L929 cells also require a certain level of JNK2 and JNK3 for survival. In contrast to the wild type JNK1, transiently expressed wild type JNK2 and JNK3 induced L929 cell death, indicating that the kinetics of activation of JNK isoforms is different.
Taken together, phosphorylation of Tyr 33 and probably Tyr 61 is important for activating WOX1. Our data support the possibility that WOX1 is a signaling protein in the p53 and JNK1 stress pathways. Whether activated JNK1 phosphorylates WOX1 remains to be determined.